Dipeptidyl peptidase I is known to release active granulocyte serine proteases of lymphatic cells from their pro-forms. It participates in mechanisms that are used physiologically by cytotoxic lymphocytes in immune defence. In the case of pathophysiological processes such as malignant transformations of myeloid and lymphatic cells, the suppression of such mechanisms can be used for the treatment of carcinomas, immune diseases or metabolic diseases. The inhibitors of DP I according to the invention can be used for the treatment of such pathophysiological conditions and diseases.
In addition to proteases involved in non-specific proteolysis, which ultimately results in the breakdown of proteins into amino acids, regulatory proteases are known which take part in the functionalisation (activation, deactivation, modulation) of endogenous peptides (Kirschke et al., 1995; Krätusslich & Wimmer, 1987). In the immunological research and neuropeptide research, a number of such so-called convertases, signal peptidases or enkephalinases have been discovered (Gomez et al., 1988; Ansorge et al., 1991).
Dipeptidyl peptidase I (DP I, Peptidase Classification Clan CA, Family C1, IUBMB Enzyme Classification EC 3.4.14.1, CAS Registration No. 9032-68-2), formerly known as cathepsin C, was discovered in 1948 by Gutman & Fruton. DP I removes dipeptides sequentially from unsubstituted N-termini of polypeptide substrates with a relatively broad substrate specificity (McDonald et al., 1971; McDonald & Schwabe, 1977). DP I is a lysosomal cysteine protease which, by removing N-terminal dipeptides, is able to release active enzymes from proenzymes, such as granzyme A, granzyme B, leucocyte elastase, cathepsin B, neuraminidase, in the lysosomal granula of cytotoxic T-lymphocytes (Kummer et al., 1996; Thiele & Lipsky, 1997).
Therefore It is commonly assumed that the DP I is involved in pathological mechanisms such as apoptotic processes, muscular dystrophy and carcinogenesis (Aoyagi et al., 1983; Gelman et al., 1980; Schlangenauff et al., 1992; Shi et al., 1992).
DP I is known as the convertase of the blood-sugar-raising hormone glucagon which, in enzymatically reduced concentration, can lead to life-threatening hypoglycaemia (McDonald, J. K. et al., 1971).
Only weak inhibition of DP I is achieved by reversible and irreversible cysteine protease-inhibitors such as leupeptin and E-64, respectively (Nikawa et al., 1992). Stronger reversible inhibitors are stefin A and chicken cystatin, protein-inhibitors from the cystatin super-family, (Nicklin & Barrett, 1984). Specific inhibition has been achieved with the a priori reactive affinity labels of the diazomethyl ketone and sulphonylmethyl ketone type (Angliker et al., 1989; Green & Shaw, 1981; Hanzlik, R. P. & Xing, R., 1998). In the last few years, other new reversible DP I-inhibitors and irreversibly acting affinity labels of DP I have become known (Palmer et al., 1998; Thiele et al., 1997).
Such reversible inhibitors, which are able to display only short-term effects caused by diffusion processes, and the affinity labels that act irreversibly on the target enzyme in vitro but which, because of their chemically reactive radical which is present a priori, are able to react, prior to their interaction with the target enzyme, with other nucleophiles and electrophiles in biological fluids. Another type, mechanism-oriented inhibitors are distinguished by becoming catalytically attacked and activated only by the target enzyme. Such inhibitors are also known as suicide inactivators. Highly efficient suicide inactivators for cysteine proteases have been developed with the class of N-peptidyl, O-acyl hydroxylamines (Brömme et al., 1996). Inhibitors of DP I have not been derived from that class of compounds since DP I is inert towards typical irreversible cysteine protease-inhibitors known in the art, such as, for example, E-64.
Furthermore, N-terminally unprotected dipeptide derivatives tend towards rapid, intramolecular decomposition.
Inhibitors of DP I are described in WO9324634; U.S. Pat. No. 5,776,718; EP0995756; DE19834610; WO0220804; EP1188765, which are incorporated herein in their entirety concerning their structure, production and use.
Other helpful references include:    Ansorge, S., Schön, E., and Kunz, D. (1991). Membrane-bound peptidases of lymphocytes: functional implications. Biomed. Biochim. Acta 50, 799-807.    Angliker, H., Wikstrom, P., Kirschke, H., and Shaw, E. (1989). The inactivation of the cysteinyl exopeptidases cathepsin H and C by affinity-labelling reagents. Biochem. J. 262, 63-68.    Aoyagi T., Wada, T., Kojima, F., Nagai, M., Miyoshino, S., and Umezawa, H. (1983). Two different modes of enzymatic changes in serum with progression of Duchenne muscular dystrophy. Clin. Chim. Acta 129, 165-173.    Brömme, D., Neumann, U., Kirschke, H., and Demuth, H. -U. (1996). Novel N-peptidyl-O-acyl hydroxamates: selective inhibitors of cysteine proteinases. Biochim. Biophys. Acta. 1202, 271-276.    Brömme, D., Demuth, H. U. (1994). N,O-Diacyl hydroxamates as selective and irreversible inhibitors of cysteine proteinases. Methods in Enzym. 244, 671-685.    Gelman B. B., Papa, L., Davis, M. H., and Gruenstein, E. (1980). Decreased lysosomal dipeptidyl aminopeptidase I activity in cultured human skin fibroblasts in Duchenne's muscular dystrophy. J. Clin. Invest. 65, 1398-1406.    Gomez, S., Gluschankof, P., Lepage, A., and Cohen, P. (1988). Relationship between endo- and exopeptidases in a processing enzyme system: activation of an endoprotease by the aminopeptidase B-like activity in somatostatin-28 convertase. Proc Natl Acad Sci USA 85, 5468-5472.    Green G. D. J. & Shaw, E. (1981). Peptidyl diazomethyl ketones are specific inactivators of thiol proteinases. J. Biol. Chem. 256, 1923-1928.    Gutman H. R. & Fruton, J. S. (1948). On the proteolytic enzymes of animal tissues VIII. An intracellular enzyme related to chymotrypsin. J. Biol. Chem. 174, 851-858.    Hanzlik, R. P. & Xing, R. (1998). Azapeptides as inhibitors and active site titrants for cysteine Proteinases. J. Med. Chem. 41, 1344-1351.    Kirschke, H., Barrett, A. J., and Rawlings, N. D. (1995). Proteinases 1: lysosomal cysteine proteinases. Protein Profile 2, 1581-1643.    Kräusslich, H.-G. and Wimmer, E. (1987). Viral Proteinases. Ann. Rev. Biochem. 57, 701    Kummer, J. A., Kamp, A. M., Citarella, F., Horrevoets, A. J. G., and Hack, C. E. (1996). Expression of human recombinant granzyme A zymogen and its activation by the cysteine proteinase cathepsin C. J. Biol. Chem. 271, 9281-9286.    McDonald, J. K., Callahan, P. X., Ellis, S., and Smith, R. E. (1971). Polypeptide degradation by dipeptidyl aminopeptidase I (cathepsin C) and related peptidases. In: Tissue Proteinases (Barrett, A. J. & Dingle, J. T., eds). Amsterdam: North-Holland Publishing, pp. 69-107.    McDonald, J. K. & Schwabe, C. (1977). Intracellular exopeptidases. In: Proteinases in mammalian cells and tissues (Barrett, A. J., ed.). Amsterdam: North Holland Publishing, pp. 311-391.    Nicklin, M. J. H. & Barrett, A. J. (1984). Inhibition of cysteine proteinases and dipeptidyl peptidase I by egg-white cystatin. Biochem. J. 223, 245-253.    Nikawa, T., Towatari, T., and Katunuma, N. (1992). Purification and characterization of cathepsin J from rat liver. Eur. J. Biochem. 204, 381-393.    Palmer, J. T., Rasnick, D., and Klaus, J. L. (1998). Reversible protease inhibitors. U.S. Pat. No. 5,776,718    Schlagenauff, B., Klessen, C., Teichmann-Dörr, S., Breuninger. H., and Rassner, G. (1992). Demonstration of proteases in basal cell carcinomas. A histochemical study using amino acid-4-methoxy-2-naphthylamides as chromogenic substrates. Cancer 70, 1133-1140.    Shi, L., Kam, C.-M., Powers, J. C., Aebersold, R., and Greenberg, A. H. (1992). Purification of three cytotoxic lymphocyte granule serine proteases that induce apoptosis through distinct substrate and target cell interactions. J. Exp. Med. 176, 1521-1529.    Thiele, D. L., Lipsky, P. E., and McGuire, M. J. (1997). Dipeptidyl Peptidase-I inhibitors and uses thereof. U.S. Pat. No. 5,602,102